No abstract

Bacterial binding protein-dependent transport systems belong to the superfamily of ABC transporters, which is widely distributed among living organisms. Their hydrophobic membrane proteins are the least characterized components. The primary structures of 61 integral membrane proteins from 35 uptake systems were compared in order to characterize a short conserved hydrophilic segment, with a consensus EAA---G---------I-LP, located approximately 100 residues from the C-terminus. Secondary structure predictions indicated that this conserved region might be formed by two amphipathic alpha-helices connected by a loop containing the invariant G residue. We classified the conserved motifs and found that membrane proteins from systems transporting structurally related substrates specifically display a greater number of identical residues in the conserved region. We determined a consensus for each class of membrane protein and showed that these can be considered as signatures.

Cells of Escherichia coli take up vitamin B 12 (cyano-cobalamin [CN-Cbl])and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella. The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His 6 tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.The outer membrane (OM) of gram-negative bacteria forms a permeability barrier which restricts passage of both nutrients and toxic environmental agents (19,25). Most nutrients cross the OM into the periplasmic space by diffusion through general or specific porins, such as OmpF or LamB. Nutrients which are too large or scarce to enter efficiently through the porins are taken into the periplasm via specific, high-affinity active transport systems. The transport systems for passage across the OM of ferric iron complexed with siderophores, heme, or host iron-binding proteins, and of cobalamins (Cbls) such as vitamin B 12 (CN-Cbl), consist of a substrate-specific TonB-dependent OM transporter, the transperiplasmic energy-coupling protein TonB, and its ancillary proteins ExbB and ExbD in the cytoplasmic membrane (CM).Most nutrients are transported across the CM by active transport systems coupled to a ...

Salmonella species are important zoonotic pathogens that cause gastrointestinal disease in humans and animals. Poultry products contaminated with these pathogens are one of the major sources of human Salmonella infections. Vaccination of chickens, along with other intervention measures, is an important strategy that is currently being used to reduce the levels of Salmonella in poultry flocks, which will ultimately lead to lower rates of human Salmonella infections. However, despite numerous studies that have been performed, there is still a need for safer, well-defined Salmonella vaccines. This review examines the different classes of Salmonella vaccines that have been tested, highlighting the merits and problems of each, and provides an insight into the future of Salmonella vaccines and the platforms that can be used for delivery.

The type III secretion system (T3SS) is an important genetic determinant that mediates interactions between Gram-negative bacteria and their eukaryotic hosts. Our understanding of the T3SS continues to expand, yet the availability of new bacterial genomes prompts questions about its diversity, distribution and evolution. Through a comprehensive survey of ∼20 000 bacterial genomes, we identified 174 non-redundant T3SSs from 109 genera and 5 phyla. Many of the bacteria are environmental strains that have not been reported to interact with eukaryotic hosts, while several species groups carry multiple T3SSs. Four ultra-conserved Microsynteny Blocks (MSBs) were defined within the T3SSs, facilitating comprehensive clustering of the T3SSs into 13 major categories, and establishing the largest diversity of T3SSs to date. We subsequently extended our search to identify type III effectors, resulting in 8740 candidate effectors. Lastly, an analysis of the key transcriptional regulators and circuits for the T3SS families revealed that low-level T3SS regulators were more conserved than higher-level regulators. This comprehensive analysis of the T3SSs and their protein effectors provides new insight into the diversity of systems used to facilitate host-bacterial interactions.